This invention relates to a human secretory type phospholipase A2; a DNA encoding this protein; a vector having the DNA; a transformant having the vector; a method for producing the protein by using the transformant; an antibody recognizing the protein; a screening method for a compound by using the protein; and a compound obtained by the screening method.
Phospholipase A2 (PLA2; EC 3.1.1.4) is a general term of phospholipids-cleaving enzymes that hydrolyze the 2-acyl ester bond of 3-sn-phosphoglyceride. PLA2 is involved in the digestion of phospholipids in food and the generation and metabolism of phospholipids in the cell membranes. In addition, PLA2 plays as a rate-limiting enzyme of the arachidonic acid cascade in the production of lipid mediators including prostaglandins. It has been known that various types of PLA2s exist in mammals. PLA2s are classified into 4 different families, such as the secretory PLA2, cytosolic PLA2, Ca2+-independent PLA2, and platelet-activating factor-acetylhydrolase, based on the localization, Ca2+ requirement, and substrate specificity (Balsinde et al., J. Biol. Chem. 272, 16069-16072 (1997)).
Among them, secretory PLA2 family comprises PLA2 enzymes that are secreted into the outside of the cells and have a relatively low molecular weight (13,000-15,000). As the member of the family, 5 types including IB type, IIA type, IIC type, V type, and X type, have been already identified. Each molecule has 12 to 16 Cys residues that form disulfide-bonds in the molecule, and possesses a consensus active site consisting of His-Asp residues. In addition, the molecules have a common Ca2+ binding region. Micro mole order of Ca2+ concentration is required for the exertion of the enzyme activity (Tischfield et al., J. Biol. Chem., 272, 17247-17250 (1997), Cupillard et al., J. Biol. Chem., 272, 15745-15752 (1997)).
It is assumed that IB type has a function as a digestive enzyme in the pancreas etc. and is involved in the progression of inflammation, such as endotoxin shock, through the binding to its specific receptor. It is also assumed that IIA type plays a role in various inflammatory responses because this type is expressed in blood platelets and synovial cells etc., and its expression is elevated during stimulation of inflammatory cytokines. However, the inflammatory response is normal in genetically IIA-deficient mice. Thus, its pathological significance remains unresolved. V type is expressed in the heat and several inflammatory cells. X type is expressed in the tissues related to the immunity such as spleen and thymus. Although it is suggested that both types are involved in the bio-regulation and inflammatory response, their importance in the body is not clearly identified (Hanasaki et al., Cell Technology, 17, 694-701 (1998)).
The object of this invention is to provide a novel type of human secretory phospholipase A2; a DNA encoding the protein of this invention; a vector comprising the DNA of this invention; a transformant having the vector of this invention; a method for producing human secretory type phospholipase A2 by using the transformant of this invention; an antibody specifically recognizing the protein of this invention; a screening method of a compound by using the protein of this invention; and a compound obtained form the screening method.
In the process of intensive studies regarding the physiological function of mouse X type PLA2, the inventors found the presence of partial sequence with homology to mouse X type PLA2 in Expressed Sequence Tags (EST) database. Based on the partial sequence, they found a DNA sequence encoding novel secretory type PLA2 protein from mouse spleen cDNA library. Further, the inventors found out a DNA sequence encoding human secretory type PLA2 protein (IID type) from human spleen cDNA library, to accomplish this invention.
The invention relates to:
A protein which comprises an amino acid sequence from First Gly to 125th Cys of that shown in SEQ ID No.:27;
The protein as described above which comprises an amino acid sequence from xe2x88x9220th Met to 125th Cys of that shown in SEQ ID No.:27;
A protein which comprises the above described amino acid sequence, in which one or more amino acid residues are substituted, deleted, inserted, or added, and has a secretory type phospholipase A2 activity;
A DNA which encodes the protein as described above;
The DNA as described above which comprises a base sequence from 89th A to 463rd C of that shown in SEQ ID No.:26;
The DNA as described above which comprises a base sequence from 29th to 463rd C of that shown in SEQ ID No.26;
A DNA which hybridizes to the DNA as described above under the stringent condition and encodes the protein having a secretory type phospholipase A2 activity;
A vector which has the DNA as described above;
A transformant which is obtained by inserting the expression vector as described above to a host;
The transformant as described above wherein the host is a mammalian cell line;
A method for producing recombinant secretory type phospholipase A2 which comprises a step for culturing the transformant as described above and a step of recovering a produced recombinant protein from the culture;
An antibody which specifically recognizes the protein as described above;
A diagnostic agent for secretory type phospholipase A2-related diseases, which comprises the antibody as described above;
An assay kit for secretory type phospholipase A2, which comprises the antibody as described above;
A therapeutic agent for secretory type phospholipase A2-related diseases, which comprises the antibody as described above;
A screening method of a compound specifically inhibiting a secretory type phospholipase A2 activity which uses the protein as described above; and
A compound which is obtained by the screening method as described above.
The protein of this invention is xe2x80x9cA protein which comprises an amino acid sequence from first Gly to 125th Cys of that shown in SEQ ID No.:27xe2x80x9d. Preferable is xe2x80x9cA protein which comprises an amino acid sequence from xe2x88x9220th Met to 125th Cys of that shown in SEQ ID No.:27xe2x80x9d. xe2x80x9cA protein which comprises an amino acid sequence from first Gly to 125th Cys of that shown in SEQ ID No.:27xe2x80x9d means a mature protein. xe2x80x9cA protein which comprises an amino acid sequence from xe2x88x9220th Met to 125th Cys of that shown in SEQ ID No.:27xe2x80x9d is an immature protein that has a signal peptide. The protein of this invention also includes xe2x80x9cA protein which comprises the amino acid sequence as described above, in which one or more amino acid residues are substituted, deleted, inserted, or added, and has a secretory type phospholipase A2 activityxe2x80x9d. Number or site of xe2x80x9csubstitution, deletion, insertion, addition of amino acid residuexe2x80x9d is not limited, if the modified protein has the same activity as the protein consisting of the amino acid sequence shown in SEQ ID No.:27. In this invention, xe2x80x9cphospholipase A2 activityxe2x80x9d means xe2x80x9cphospholipid-cleaving activity that hydrolyzes 2-acyl ester bond of 3-sn-phosphoglyceride in a Ca2+-dependent mannerxe2x80x9d.
Although these mutations in the amino acid sequences can be caused naturally by mutation or modification after transcription, artificial modification can also be caused by the DNA of this invention. The protein of this invention includes all proteins which are encoded by modified DNA have the characteristics as mentioned above regardless of the cause or mean of these modification/mutation.
The DNA of this invention means xe2x80x9ca DNA encoding the protein of this inventionxe2x80x9d. As the DNA of this invention, a DNA encodes the mature protein and comprises a base sequence from 89th G to 463rd C of that shown in SEQ ID No.:26 is preferably given for example. More preferably, a DNA encodes the immature protein and comprises a base sequence from 29th A to 463rd C of that shown in SEQ ID No.:26 is given for example. A DNA that hybridizes to the DNA of this invention under the stringent condition and encodes the protein having a secretory type phospholipase A2 activity is also included in the DNA of this invention. xe2x80x9cA DNA that hybridizes to the DNA of this invention under the stringent conditionxe2x80x9d can be obtained by using the DNA of the encoding region as a probe. xe2x80x9cHybridize under the stringent conditionxe2x80x9d means that positive hybridizing signal can be observed after heating at 42xc2x0 C. in 6xc3x97SSC, 0.5% SDS and 50% formamide solution and washing 68xc2x0 C. in 0.1xc3x97SSC, 0.5% SDS solution.
Using the DNA of this invention, a production of the recombinant protein can be performed based on textbooks and references such as Molecular Cloning etc. More concretely, a transcription initiation codon is added at upper stream of the DNA to be expressed, and a transcription stop codon is added at down stream of the DNA. A regulator gene such as a promoter sequence (Ex. Trp, lac, T7, SV40 initial promoter) which controls transcription is also added. The expression plasmid, which can replicate and work in the host cells, is prepared by insertion of the DNA into an appropriate vector (ex. PBR322, pUC19, pSV.SPORT1 etc.).
The transformant is obtained by insertion of the expression vector into host cells. As a host cell, procaryotes such as E. coli, monocellular eukaryotes such as yeast, and cells derived from multicellular organisms such as insects and mammals are given for examples. Mammal""s cells are preferable. As a mammal""s cell, CHO cell, 293 cell, COS-7 cell are given for example.
The antibody of this invention is an antibody against the polypeptide of this invention or peptide fragment that can compose the epitope, and includes both of polyclonal antibodies and monoclonal antibodies. Secretory type PLA2 is involved in the release of fatty acids (ex. arachidonic acid). Excess release of fatty acids cause various diseases such as septic shock, adult respiratory distress syndrome, pancreatitis, bronchial asthma, allergic rhinitis, rheumatoid arthritis, etc. It is possible to diagnose these diseases by the evaluation of a concentration of secretory type phospholipase A2. The antibody of this invention provides a diagnostic agent and an assay kit for secretory type phospholipase A2-related diseases. If the antibody inhibits a PLA2 activity, the antibody itself can be a therapeutic agent for the diseases caused by PLA2.